CN114350846B

CN114350846B - Primer group and kit for combined detection of multiple pulmonary infection fungi

Info

Publication number: CN114350846B
Application number: CN202210093716.5A
Authority: CN
Inventors: 张祥林; 张劲松; 侯艳雯; 魏鹏; 陈晨; 胡秋萍
Original assignee: Chongqing Pasteur Biomedical Technology Co ltd
Current assignee: Chongqing Pasteur Biomedical Technology Co ltd
Priority date: 2022-01-26
Filing date: 2022-01-26
Publication date: 2024-03-15
Anticipated expiration: 2042-01-26
Also published as: CN114350846A

Abstract

The invention relates to the technical field of biology, in particular to a primer set and a kit for jointly detecting various pulmonary infection fungi, wherein the PCR primer set comprises a candida albicans detection primer pair, a candida tropicalis detection primer pair, a candida glabrata detection primer pair, an aspergillus fumigatus detection primer pair, an aspergillus flavus detection primer pair, a aspergillus niger detection primer pair and a novel cryptococcus detection primer pair. The kit comprises multiple PCR reactants, wherein the multiple PCR reactants comprise the PCR primer group. The primer group and the kit can be used for jointly detecting seven pulmonary infection fungi by combining multiple PCR and a capillary fragment analysis method, and the method has the advantages of simple operation, accurate detection, high flux, high sensitivity and high specificity, obviously shortens the detection time, and can realize the detection result within 2-3 hours.

Description

Primer group and kit for combined detection of multiple pulmonary infection fungi

Technical Field

The invention relates to the technical field of biology, in particular to a primer group and a kit for jointly detecting various pulmonary infection fungi.

Background

Fungi are widely distributed in natural environment, are common bacteria colonized on the surfaces of human skin and mucous membranes, and are symbiotic with human beings by avoiding the human immune system. However, in the event of an impaired immune system or a disrupted host barrier, fungi can invade the human body, leading to a lethal infection. Invasive pulmonary fungal infections (Invasive Pulmonary Fungal Infection, IPFI) are bronchopulmonary fungal infections that do not include fungal parasitism and allergies, and are divided into primary and secondary types. In recent years, with aging of population, use of immunosuppressants for organ transplantation, radiotherapy and chemotherapy of tumors, hematopoietic stem cell transplantation, combination of ultra-broad-spectrum antibiotics and various antibiotics, use of corticosteroid hormones, various catheter interventions and the like, the incidence of invasive pulmonary fungal infection is gradually increased. Common fungi responsible for invasive pulmonary fungal infections are mainly candida, aspergillus, cryptococcus, zygomycetes (mainly mucor), pneumosporosis, and the like. Because the clinical manifestation of the pulmonary fungal infection is often not specific, early diagnosis is difficult, and the disease condition is easily covered by basic diseases, misdiagnosis and missed diagnosis are caused, and treatment is delayed. The accurate and timely diagnosis of pulmonary fungal infections depends on laboratory accurate detection results.

Common laboratory methods for detecting pulmonary fungal infections include: direct microscopy, histopathological examination, fungal culture, serological detection (1, 3-beta-D glucan detection, galactomannan detection, cryptococcus capsular polysaccharide antigen detection, aspergillosis antibodies, etc.), imaging examination, and nucleic acid detection-based molecular biological methods, and the like. Currently, histopathological examination remains the "gold standard" for diagnosing fungal infections. But is intolerable to the patient because it is an invasive procedure. The result is limited by the accuracy of the materials, and the positive rate is low. The diagnosis result is subjected to a series of experimental processes, which consumes longer time and is unfavorable for early diagnosis. Direct microscopy is the most classical method and has the characteristics of rapidness, simplicity and convenience, but the positive rate is lower. Traditional fungal culture methods require at least 3-5 days, even longer, and are less sensitive, which is detrimental to early diagnosis. The antigen detection specificity is better, but the sensitivity is lower, and false negative is often caused. Antibody detection has limited significance for early diagnosis of invasive pulmonary fungal infections. Molecular biology methods represented by fluorescent quantitative PCR techniques have gradually become an important method for diagnosing invasive pulmonary fungal infection. Although fluorescent quantitative PCR has high specificity, sensitivity and timeliness, there are also the following disadvantages: (1) low flux: limited by the fluorescent channel, single tubes can only detect 3 fungi; (2) high cost: and multiple fungi are detected simultaneously, so that the cost is high.

Disclosure of Invention

In view of the above drawbacks of the prior art, the present invention is directed to a primer set and a kit for combined detection of various pulmonary infectious fungi, which are used for solving the problems in the prior art.

To achieve the above and other related objects, the present invention provides a PCR primer set for the joint detection of a plurality of pulmonary infection fungi, the PCR primer set including a candida albicans detection primer pair, a candida tropicalis detection primer pair, a candida glabrata detection primer pair, an aspergillus fumigatus detection primer pair, an aspergillus niger detection primer pair, a novel cryptococcus detection primer pair.

Preferably, the PCR primer set further comprises the following features:

1) Candida albicans detection primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2;

2) Candida tropicalis detection primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.3 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 4;

3) Candida glabrata detection primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.5 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 6;

4) Aspergillus fumigatus detection primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.7 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 8;

5) Aspergillus flavus detection primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.9 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 10;

6) Aspergillus niger detection primer pair: comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.11 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 12;

7) Novel cryptococcus detection primer pair: comprising an upstream primer with a nucleotide sequence shown as SEQ ID NO.13 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 14.

The invention also provides application of the PCR primer group in preparation of a kit for detecting candida albicans, candida tropicalis, candida glabrata, aspergillus fumigatus, aspergillus flavus, aspergillus niger and novel cryptococcus.

The invention also provides a kit for jointly detecting candida albicans, candida tropicalis, candida glabrata, aspergillus fumigatus, aspergillus flavus, aspergillus niger and novel cryptococcus, wherein the kit comprises multiple PCR reactants, and the multiple PCR reactants comprise the PCR primer group.

As described above, the primer group and the kit for the joint detection of various pulmonary infection fungi have the following beneficial effects: seven pulmonary infection fungi can be detected or identified in a combined way, and the method has the advantages of simple operation, accurate detection, high flux, high sensitivity and high specificity, obviously shortens the detection time, and can realize the detection result within 2-3 hours. The human DNA internal reference can ensure the quality control of the nucleic acid in the detection process of nucleic acid extraction and PCR amplification, and avoid the problem of false negative caused by the problems of nucleic acid extraction quality and PCR amplification. Multiple PCR and capillary fragment analysis can be combined to realize single tube detection of various pulmonary infection fungi, ensure the detection flux and have lower cost, and are suitable for disease control centers, hospitals and other medical institutions.

Drawings

FIG. 1 is a graph showing the results of capillary electrophoresis separation of Candida albicans in example 2 of the present invention.

FIG. 2 is a graph showing the results of capillary electrophoresis separation of Cryptococcus neoformans in example 2 of the present invention.

FIG. 3 is a graph showing the results of capillary electrophoresis separation of positive quality control in example 2 of the present invention.

FIG. 4 shows the results of the specific analysis in example 3 of the present invention.

FIG. 5 shows the amplification effect of primer set 1 in comparative example 2 of the present invention.

FIG. 6 shows the amplification effect of primer set 2 in comparative example 2 of the present invention.

FIG. 7 shows the amplification effect of primer set 3 in comparative example 2 of the present invention.

Detailed Description

The invention provides a PCR primer group for detecting pulmonary infection fungi, which comprises the following primer pairs:

In the present invention, the Cryptococcus neoformans are selected from Cryptococcus neoformans and/or Cryptococcus neoformans. The novel cryptococcus is a yeast with a membrane, and is divided into A, B, C, D serotypes according to polysaccharide on the capsule of the yeast, and the four serotypes correspond to the griubi variant (A), the Grignard variant (B) and the neonatal variant (D) of the novel cryptococcus respectively.

The specific nucleotide sequence of the primer set described above may be such that 1 or more nucleotides are replaced with other nucleotides, or 1 or more nucleotides may be added to the 3 'or 5' end, as long as the specific recognition regions can be recognized specifically under PCR conditions (preferably, annealing and self-annealing do not occur between the primers used in a single reaction vessel). Here, the number of the elements is, for example, 2 to 3. In the case of adding 1 or more bases to the primer, it is preferable to add to the 5' -end of the primer.

The identity between the base sequence obtained by substituting 1 or more bases in a specific base sequence of the primer pair with other bases and the base sequence before substitution (i.e., the base sequence shown in the sequence number) is preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, and more preferably 95% or more.

The length of each primer is not particularly limited as long as the corresponding specific recognition region can be specifically recognized and hybridization between primers does not occur, and is preferably 15 bases or more and 40 bases or less. More preferably, the lower limit of the length of the primer is 16 bases or more, still more preferably 17 bases or more, and still more preferably 18 bases or more. More preferably, the upper limit of the length of the primer is 39 bases or less, still more preferably 38 bases or less, and still more preferably 37 bases or less.

The PCR primer group is a multiplex PCR primer group. Each detection primer pair can be packaged independently or mixed to prepare a multiplex PCR mixture. The amount of each primer pair in the multiplex PCR detection mixture may be a conventional amount known to those skilled in the art.

Further, the use is in the preparation of a kit for the combined detection of candida albicans, candida tropicalis, candida glabrata, aspergillus fumigatus, aspergillus flavus, aspergillus niger and cryptococcus neoformans.

Further, the use is in the preparation of a kit for the combined detection of pulmonary infection with candida albicans, candida tropicalis, candida glabrata, aspergillus fumigatus, aspergillus flavus, aspergillus niger and novel cryptococcus.

The kit of the invention adopts a multiplex PCR technology to detect the seven pulmonary infection fungi simultaneously in a single tube, and can analyze and judge the infection conditions of candida albicans, candida tropicalis, candida glabrata, aspergillus fumigatus, aspergillus flavus and aspergillus niger and novel cryptococcus (new born variety/Grignard variety) according to the amplification and detection conditions. The design of the primers is critical to the kits of the invention.

Other reagents conventional for PCR may also be included in the multiplex PCR reaction, such as: multi DNA Polymerase. Because such PCR reagents commonly used are available individually or by themselves via a market approach, which reagents are specifically required to be assembled into the kit may be configured according to actual needs of the user, or may be assembled into the kit entirely for convenience.

Further, the multiplex PCR reaction also comprises an internal reference amplification primer pair. In one embodiment, the internal reference amplification primer pair is a human DNA internal reference amplification primer pair. The human DNA internal reference amplification primer pair comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.15 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 16.

The multiplex PCR reaction can be configured by itself or can be obtained by directly mixing a commercially available general PCR reaction solution without primers with the primer set. For example, the universal PCR reaction solution may be obtained by adding the primer set and the internal reference amplification primer set of the present invention to Multiplex PCR Mix (UDG) of Suzhou offshore protein technologies Co., ltd. In one embodiment, the concentration of each primer in the multiplex PCR reaction is from 100nmol/L to 300nmol/L based on the total volume of the multiplex PCR reaction.

In one embodiment, the kit further comprises a sample genome extraction reagent. The sample genome extraction reagent may be commercially available or self-formulated.

Further, the kit may further comprise a positive quality control. The positive quality control product is pUC57 plasmid DNA containing different target fungus specific nucleic acid fragments and human DNA internal reference specific nucleic acid fragments. In one embodiment, the nucleotide sequence of the positive quality control is shown as SEQ ID NO. 17-SEQ ID NO.23. Can be purchased separately or constructed by itself according to the prior art.

Further, the kit also contains a negative quality control. In one embodiment, the negative quality control is TE buffer. Either commercially available alone or self-configurable according to the prior art.

The invention also provides a use method of the kit, which comprises the following steps:

(1) After the sample is pretreated, extracting genomic DNA of the sample;

(2) Sample adding: respectively adding sample DNA, positive quality control product or negative quality control product into PCR tubes filled with the multiple PCR reactants to obtain corresponding sample reaction tubes, positive reaction tubes or negative reaction tubes;

(3) And (3) PCR reaction: the reaction tube is arranged on a PCR instrument, and circulation parameters are set for carrying out PCR reaction;

(4) After the completion of the PCR reaction, the results were analyzed.

In step (1), the extraction of genomic DNA from a sample is known in the art.

In one embodiment, in step (3), the conditions of the PCR reaction are set to: cycling for 1 time at 50 ℃ for 2 min; cycling for 1 time at 95 ℃ for 2 min; 94 ℃ for 20sec, 60 ℃ for 20sec, 72 ℃ for 30sec, and 35 times of circulation; cycling for 1 time at 72 ℃ for 5 min; 4 ℃ infinity cycle 1 time.

The invention also provides application of the kit in preparing a pulmonary infection fungus detection product.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Example 1 preparation and methods of use of the kit

The nucleotide sequences of the candida albicans detection primer pair, candida tropicalis detection primer pair, candida glabrata detection primer pair, aspergillus fumigatus detection primer pair, aspergillus flavus detection primer pair, novel cryptococcus (new born variety/gerte variety) detection primer pair are respectively synthesized as shown in the following table 1.

The kit also contains a human DNA internal reference amplification primer pair, and the nucleotide sequence of the kit is shown in the following table 1.

The kit also contains a positive quality control. The positive quality control product is pUC57 plasmid DNA containing target fungus specific nucleic acid fragments and human DNA internal reference specific nucleic acid fragments, and the nucleotide sequences are shown in Table 2.

The kit also contains a negative quality control. The negative quality control product is TE buffer solution.

The kit also comprises Multiplex PCR Mix (UDG) (product number E024-YBBB, suzhou offshore protein technology Co., ltd.) and a bacterial and fungal DNA extraction kit (product number T07-100, shanghai Meiji-Su-Hua biomedical technology Co., ltd.).

TABLE 1

TABLE 2

Example 2 evaluation of detection Effect of kit

The embodiment of the invention provides a specific use mode of the kit, each detection primer pair shown in the sequence in Table 1 and the human DNA internal reference amplification primer pair are uniformly mixed with Multiplex PCR Mix (UDG) purchased from Suzhou coastal protein technologies and technologies, inc. to obtain a multiplex PCR reactant, wherein the concentration of each primer in the multiplex PCR reactant is shown in Table 3.

TABLE 3 Table 3

1. Patient samples (sputum, bronchoalveolar lavage, etc.) were collected following standard procedures, and immediately after collection, were placed in an ice bag and immediately sent for examination.

2. And after the sample is subjected to pretreatment, extracting DNA in the sample together to obtain a DNA sample to be detected.

3. Preparation and detection of PCR reaction system

(1) Setting typesetting mode according to detection requirement, adding multiple PCR reactants into 8-joint tube or 96-well plate according to typesetting, adding 17.5 μl each well, sequentially adding 2.5 μl negative quality control (TE buffer), DNA sample to be detected and positive quality control (pUC 57 plasmid DNA containing target fungus specific nucleic acid fragment and human DNA internal reference specific nucleic acid fragment) into the reaction well, and covering.

(2) Thoroughly mixed and centrifuged.

(3) The prepared reagent reacts.

4. Setting PCR program

(1) The PCR procedure was set up according to the instructions of the PCR apparatus, and the amplification conditions for the PCR reaction are shown in Table 4 below.

TABLE 4 Table 4

(2) After the PCR amplification, the amplification plate/tube was centrifuged briefly, and then the tube cap was carefully opened, sealed with mineral oil, and detected using a nucleic acid fragment analyzer.

5. Analysis of results

And judging the sample detection result by detecting the fragment size of the reaction product. The negative control should be free of reaction product fragments. Otherwise, the experimental result is invalid and the detection is repeated. In the cationic quality control detection reaction system, a reaction product fragment exists in the range of 70-77bp and shown in the following table 5, otherwise, the experimental result is invalid and the detection is carried out again. For a sample detection reaction system, an internal reference reaction product fragment should exist within the range of 70-77bp, otherwise, the experimental result is invalid and the detection is carried out again. If the sample detection result is positive, judging the type of the infected fungi according to the signal intensity of the reaction product and the size of the corresponding fragment. The whole detection procedure is about 2-3 hours.

Referring to the experimental test data shown in fig. 1 to 3, the fragment size values corresponding to each fungus are shown in table 5 below.

TABLE 5

Fungal species	Fragment size
		Candida tropicalis	102-113bp
Candida albicans	127-138bp
		Aspergillus fumigatus	196-207bp
Candida glabrata (C.glabrata)	246-258bp
		New cryptococcus (New variety/Grignard variety)	302-312bp
Aspergillus flavus	349-359bp
		Aspergillus niger	405-415bp
Human DNA internal reference	70-77bp

Example 3 sensitivity and specificity analysis of the kit

Sensitivity analysis:

all fungal strains were purchased from the Guangdong province microorganism strain collection and the China medical culture Collection (Table 6). To determine the sensitivity of the kit, nucleic acids of Candida albicans, candida tropicalis, candida glabrata, aspergillus fumigatus, aspergillus flavus, aspergillus niger, novel Cryptococcus neoformans and novel Cryptococcus garter varieties were each subjected to a gradient dilution at a concentration of 1X 10, respectively ⁶ copies/mL、5×10 ⁵ copies/mL、1×10 ⁵ copies/mL、5×10 ⁴ copies/mL、1×10 ⁴ copies/mL、5×10 ³ copies/mL、1×10 ³ copies/mL、5×10 ² copies/mL、1×10 ² The samples were repeated 3-5 times per each gradient dilution, and multiplex PCR amplification and fragment analysis detection were performed using the same defined seven mycotoxin multiplex PCR detection system as in example 2 until no signal was detected, with each repeated 20 times, with a positive detection rate level of 95% as the lowest detection Limit (LOD), i.e., sensitivity.

The relevant pathogenic strains were as follows:

TABLE 6

The detection sensitivity of the kit of the invention is shown in the following table:

TABLE 7

Detection index	LOD(copies/mL)
		Candida albicans	1000
Candida tropicalis	1000
		Candida glabrata (C.glabrata)	1000
Aspergillus fumigatus	1000
		Aspergillus flavus	1000
Aspergillus niger	1000
		New novel Cryptococcus neoformans	5000
New cryptococcus garter variety	5000

Specificity analysis:

the specificity of the detection method established by the invention mainly shows the specificity of the specific primer. The designed primers are subjected to primer-blast comparison and analysis, have high conservation and specificity, and can specifically distinguish seven fungi. In order to determine the specificity of the kit, sixteen unrelated pathogenic strains are selected as simulated interference samples, all pathogenic strains are purchased from the Guangdong province microorganism strain collection center and the China medical culture collection center, the different pathogenic strains are detected after nucleic acid extraction, the concentration is 10 ng/mu L, the total nucleic acid of each sample obtained above is mixed with an equivalent amount of human internal reference plasmid (pUC 57) as a template for multiplex PCR amplification and fragment analysis, and the specificity of the primer design of the kit is verified.

The relevant pathogenic strains were as follows:

TABLE 8

As a result, as shown in FIG. 4, multiplex PCR and fragment analysis of the total nucleic acids of sixteen unrelated pathogens with the same amount of the human internal reference plasmid (pUC 57) as a template can amplify only the 76bp human internal reference band without other amplified bands. From the data, the detection results of the kit provided by the invention on the microorganisms are negative, and the fact that the kit provided by the invention has no cross reaction with other microorganisms is proved, so that the kit is high in pathogen detection specificity.

Example 4 application of the kit to clinical sample testing

The clinical microbiological laboratory culture detection method is selected as a reference method, and the clinical specimens of sputum and bronchoalveolar lavage fluid are detected by applying the embodiment 2 of the invention and the first generation sequencing technology. The test is used for detecting 20 samples of sputum and bronchoalveolar lavage fluid of a patient suspected to be/diagnosed with pulmonary fungal infection. The results of the sample testing are shown in Table 9 below.

TABLE 9

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According to the detection data, the detection result of the method has higher coincidence with the detection result of the culture detection method.

Comparative example 1

Comparative primer sets 1-8 were prepared as in example 1, see Table 10 below.

Table 10

The difference was only that the primers shown in SEQ ID NOS.1-2 in the primer set of example 1 were replaced with the primers shown in SEQ ID NOS.24-25 to obtain a comparative primer set 1. A comparative primer set 2 was obtained by replacing the primers shown in SEQ ID NOS.3-4 in the primer set of example 1 with the primers shown in SEQ ID NOS.26-27. A comparative primer set 3 was obtained by replacing the primers shown in SEQ ID NOS.5-6 in the primer set of example 1 with the primers shown in SEQ ID NOS.28-29. A comparative primer set 4 was obtained by replacing the primers shown in SEQ ID NOS.7-8 in the primer set of example 1 with the primers shown in SEQ ID NOS.30-31. A comparative primer set 5 was obtained by replacing the primers shown in SEQ ID NOS.9-10 in the primer set of example 1 with the primers shown in SEQ ID NOS.32-33. A comparative primer set 6 was obtained by replacing the primers shown in SEQ ID NOS.11-12 in the primer set of example 1 with the primers shown in SEQ ID NOS.34-35. A comparative primer set 7 was obtained by replacing the primers shown in SEQ ID NOS.13-14 in the primer set of example 1 with the primers shown in SEQ ID NOS.36-37. A comparative primer set 8 was obtained by replacing the primers shown in SEQ ID NOS.15-16 in the primer set of example 1 with the primers shown in SEQ ID NOS.38-39.

Minimum detection limit verification the minimum detection limit verification was performed according to the method of example 3. The lowest limit of detection for example 3 compared with the comparative example is shown in table 11 below.

TABLE 11

Detection index	Example 3 LOD (copies/mL)	Comparative example, LOD (copies/mL)
			Candida albicans	1000	1000
Candida tropicalis	1000	5000
			Candida glabrata (C.glabrata)	1000	5000
Aspergillus fumigatus	1000	1000
			Aspergillus flavus	1000	5000
Aspergillus niger	1000	1000
			New novel Cryptococcus neoformans	5000	5000
New cryptococcus garter variety	5000	5000

As can be seen from Table 11, the kit of the present disclosure has a stronger detection ability than the comparative examples for trace amounts of nucleic acids of Candida glabrata, candida tropicalis, aspergillus flavus in the sample.

Specificity verification was performed according to the method of example 3. The results showed that the results of the reactions for the primer pairs of the comparative examples were all negative.

As can be seen from the comparison of the example 3 and the comparative example, seven pulmonary infection fungi can be detected at one time, the sensitivity is high, the specificity is high, the minimum detection limit is lower, and the coverage is wider.

Comparative example 2

This comparative example, using candida glabrata as an example, demonstrates primers that were found to have an undesirable effect in part during the development process. Candida glabrata primer sequences and screening: for the 3 groups of primer combinations, the primer amplification effect is firstly screened by single PCR amplification, and the single detection result shows that the primer pair 3 has lower amplification efficiency, and the primer pairs 1 and 2 can basically meet the requirement of subsequent experiments (as shown in figures 5 to 7) and need to be added into a multiplex PCR method for further verification. The primer pairs 1 and 2 are respectively added into a multiplex PCR system for amplification, and the detection results are as follows:

primer pair 1

F-1：(SEQ ID NO.40)ACTATTCTTTTGTTCGTTGGGG

R-1：(SEQ ID NO.41)CAATGTGCGTTCAAAGATTCG

Primer pair 2

F-2：(SEQ ID NO.42)TCGATGAAGAACGCAGCGAAATGCG

R-2：(SEQ ID NO.43)CGCAAACGAGCAGCAGATTA

Primer pair 3

F-3：(SEQ ID NO.44)ACATTGCGCCCTCTGGTA

R-3：(SEQ ID NO.45)CAAAACACTCACTTATCCCTCC

Primer pair 1 was added to multiplex PCR system detection results: primer pair 1 causes the amplification efficiency of candida albicans to be low, and the primer pair 1 and candida albicans primer pair may have competitive inhibition or primer dimer to cause the amplification efficiency of the primer pair to be reduced, thereby influencing the requirement of subsequent detection. Primer pair 2 was added to multiplex PCR system detection results: the amplification efficiency of each primer pair was essentially unchanged. From comprehensive consideration of multiple aspects, the primer pair 2 is selected as a primer pair of candida glabrata in a multiplex PCR detection system, and the system is repeatedly verified to meet the detection requirement.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

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<213> Artificial sequence (Artificial Sequence)

<400> 18

tggaagttaa aaaagcgtaa caaggtttcc gtaggtgaac ctgcggaagg atcattactg 60

atttgcttaa ttgcaccaca tgtgtttttt attgaacaaa tttctttggt ggcgggagca 120

atcctaccgc cagaggttat aactaaacca aactttttat ttacagtcaa acttgattta 180

ttattacaat agtcaaaact ttcaacaacg gatctcttgg ttctcgcatc gatgaagaac 240

gcagcgaaat gcgatacgta atatgaattg cagatattcg tgaatcatcg aatctttgaa 300

cgcacattgc gccctttggt attccaaagg gcatgcctgt ttgagcgtca tttctccctc 360

aaacccccgg gtttggtgtt gagcaatacg ctaggtttgt ttgaaagaat ttaacgtgga 420

aacttatttt aagcgactta ggtttatcca aaaacgctta ttttgctagt ggcca 475

<210> 19

<211> 578

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

actactattc ttttgttcgt tgggggaaag ctctctttcg ggagggagtt ctcccagtgg 60

atgcaaacac aaacaaatat ttttttaaaa taattcagtc aacacaagat ttcttttagt 120

agaaaacaac ttcaaaactt tcaacaatgg atctcttggt tctcgcatcg atgaagaacg 180

cagcgaaatg cgatacgtaa tgtgaattgc agaattccgt gaatcatcga atctttgaac 240

gcacattgcg ccctctggta ttccgggggg catgcctgtt tgagcgtcat ttccttctca 300

aacacattgt gtttggtagt gagtgatact ctcgtttttg agttaacttg aaattgtagg 360

ccatatcagt atgtgggaca cgagcgcaag cttctctatt aatctgctgc tcgtttgcgc 420

gagcggcggg ggttaatact gtattaggtt ttaccaactc ggtgttgatc tagggaggga 480

taagtgagtg ttttgtgcgt gctgggcaga cagacgtctt taagtttgac ctcaaatcag 540

gtagggttac ccgctgaact taagcatatc aataagcg 578

<210> 20

<211> 589

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

ctgcggaagg atcattaccg agtgagggcc ctctgggtcc aacctcccac ccgtgtctat 60

cgtaccttgt tgcttcggcg ggcccgccgt ttcgacggcc gccggggagg ccttgcgccc 120

ccgggcccgc gcccgccgaa gaccccaaca tgaacgctgt tctgaaagta tgcagtctga 180

gttgattatc gtaatcagtt aaaactttca acaacggatc tcttggttcc ggcatcgatg 240

aagaacgcag cgaaatgcga taagtaatgt gaattgcaga attcagtgaa tcatcgagtc 300

tttgaacgca cattgcgccc cctggtattc cggggggcat gcctgtccga gcgtcattgc 360

tgccctcaag cacggcttgt gtgttgggcc cccgtccccc tctcccgggg gacgggcccg 420

aaaggcagcg gcggcaccgc gtccggtcct cgagcgtatg gggctttgtc acctgctctg 480

taggcccggc cggcgccagc cgacacccaa ctttattttt ctaaggttga cctcggatca 540

ggtagggata cccgctgaac ttaagcatat caataggcca ggagaggaa 589

<210> 21

<211> 701

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

actcaacgct gtgcgccgat acacttctca cactgctact tctacgactc cccccacctc 60

gccattcgct cctcgccatt ttctttccat cgctgatctt acctcaacgg agttcgcaac 120

cctcgtccgt aatgcatctt cacacaaacg aaccatcaag tcgggatcaa tccctcagaa 180

cctgcttggt tctatgaccg gacagactgt agctatgctg ttcagcaaac gcagtaccag 240

aaccaggata tctactgaag gggctgtggt gcgcttggga ggacatccaa tgtttctggg 300

caaggatgat attcaactcg gtgtcaatga gtccttgtat gactctgcgg ttgtaatctc 360

ctccatggtc tcttgtattg tagcccgagt cggcaagcac gctgaggttg cagaccttgc 420

caagcattca accgtgcctg ttatcaacgc tctctgtgac tccttccacc ctctccaggc 480

cattgccgat tttcagacca tctacgaaac atttacacct aaggcccatc gctctgacag 540

tttgggtctg gagggcctca agattgcctg ggtcggtgat gcaaacaacg ttctgttcga 600

catggcaatt gctgctacca agatgggtat tgatatcgcc gttgcgactc cgaagggcta 660

tgagattcct gccccgatgt tggagctcat caagcaggcc a 701

<210> 22

<211> 700

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

ccgatattat cacagctact ttgtctaaaa gtgttggctg cactggagga ttcgtcgccg 60

caaatggtat ctgtgcccaa cagcttagac ttcaagatga gctgttatca cacgaagggg 120

cagaatcttt gtccacggta gctcttgtac ggactttaag cctgctgaaa aagacccgtt 180

tgattgagta tcgcatgcgt caattgaagg ccaaagcggg atttgtgctc cagagactca 240

cagaagcagg atgcaaggtc ctctcctctc cagattcagc cattatatgt tttcctgtcg 300

gtgagttgca gtcccactcg agattatttc gaagaacctt catgctcata acacccagga 360

actgttcgac aggcgtcaat gtttcatgca gaggccctca agaggggctt tgccgtagcc 420

tgtggtgtgc caccagctac acctctttgg taagcttcac aaatgttgct taaggaatct 480

gctgacggaa cagggcctgt cgaattcgaa tgtgcgtgtt tgctacgtcc tcctgggctg 540

acatcctgaa tctggtcaac gcaacgatct cagtggcttg caaactaaat atccatggga 600

tcaaacccat ggtattggat gagagctgcc ttccttacga tagtggggag ccgctagatg 660

tagctgcaga gagtgaggcg acagataaag agtttcatga 700

<210> 23

<211> 472

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

aaggatcagc agagaatatt ggacttcggg tccatttatc tacccatcta cacctgtgaa 60

ctgtttatgt gcttcggcac gttttacaca aacttctaaa tgtaatgaat gtaatcttat 120

tataacaata ataaaacttt caacaacgga tctcttggct tccacatcga tgaagaacgc 180

agcgaaatgc gataagtaat gtgaattgca gaattcagtg aatcatcgaa tctttgaacg 240

caacttgcgc cctttggtat tccgaagggc atgcctgttt gagagtcatg aaaatctcaa 300

tccctcgggt tttattacct gttggacttg gatttgggtg tttgccgcga cctgcaaagg 360

acgtcggctc gccttaaatg tgttagtggg aaggtgatta cctgtcagcc cggcgtaata 420

agtttcgctg ggcctatggg gtagtcttcg gcttgctgat aacaaccatc tc 472

<210> 24

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

tgaagaacgc agcgaaatg 19

<210> 25

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

cagcggtttg agggagaaa 19

<210> 26

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

gaatcatcga atctttgaac gc 22

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

tattgctcaa caccaaaccc 20

<210> 28

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

tcgatgaaga acgcagcgaa atgcg 25

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

cgcaaacgag cagcagatta 20

<210> 30

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

cccgtgtcta tcgtaccttg t 21

<210> 31

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

tttcgctgcg ttcttcatc 19

<210> 32

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

tgtttctggg caaggatga 19

<210> 33

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

gagtcgcaac ggcgatat 18

<210> 34

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

gtcgccgcaa atggtatc 18

<210> 35

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

accaaagagg tgtagctggt g 21

<210> 36

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

acccatctac acctgtgaac tg 22

<210> 37

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 37

gcggcaaaca cccaaatc 18

<210> 38

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 38

cattgccctc aacgacca 18

<210> 39

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 39

ccaccaccct gttgctgta 19

<210> 40

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 40

actattcttt tgttcgttgg gg 22

<210> 41

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 41

caatgtgcgt tcaaagattc g 21

<210> 42

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 42

tcgatgaaga acgcagcgaa atgcg 25

<210> 43

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 43

cgcaaacgag cagcagatta 20

<210> 44

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 44

acattgcgcc ctctggta 18

<210> 45

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 45

caaaacactc acttatccct cc 22

Claims

1. The PCR primer set is characterized by comprising a candida albicans detection primer pair, a candida tropicalis detection primer pair, a candida glabrata detection primer pair, an aspergillus fumigatus detection primer pair, an aspergillus flavus detection primer pair, an aspergillus niger detection primer pair and a novel cryptococcus detection primer pair;

1) Candida albicans detection primer pair: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2;

2) Candida tropicalis detection primer pair: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4;

3) Candida glabrata detection primer pair: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 6;

4) Aspergillus fumigatus detection primer pair: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 8;

5) Aspergillus flavus detection primer pair: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 10;

6) Aspergillus niger detection primer pair: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.11, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 12;

7) Novel cryptococcus detection primer pair: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.13, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 14.

2. Use of the PCR primer set of claim 1 in the preparation of a kit for detecting candida albicans, candida tropicalis, candida glabrata, aspergillus fumigatus, aspergillus flavus, aspergillus niger and cryptococcus neoformans.

3. A kit for the combined detection of candida albicans, candida tropicalis, candida glabrata, aspergillus fumigatus, aspergillus flavus, aspergillus niger and cryptococcus neoformans, wherein the kit comprises a multiplex PCR reaction product comprising the PCR primer set of claim 1.

4. The kit of claim 3, wherein Multiplex PCRMix and/or internal reference amplification primer pairs are also included in the Multiplex PCR reaction.

5. The kit according to claim 4, wherein the internal reference amplification primer pair comprises an upstream primer having a nucleotide sequence shown in SEQ ID NO.15 and a downstream primer having a nucleotide sequence shown in SEQ ID NO. 16.

6. The kit according to claim 4, wherein the concentration of each primer is 100nmol/L to 300nmol/L based on the total volume of the multiplex PCR reaction.

7. The kit according to claim 3, further comprising any one or more of a sample genome extraction reagent, a positive quality control, and a negative quality control.

8. The kit according to claim 7, wherein the positive quality control is pUC57 plasmid DNA containing each target fungus specific nucleic acid fragment and human DNA reference specific nucleic acid fragment, and/or the negative quality control is TE buffer.

9. The kit according to claim 8, wherein the nucleotide sequence of the positive quality control is shown in SEQ ID NO. 17-SEQ ID NO.23.